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Initiation and regulation of fibrinolysis in human plasma at the
plasminogen activator level
TC Wun and A Capuano
The initiation and regulation of fibrinolysis has been studied by
reconstitution of fibrinolytic activity in human plasma in vitro. Depletion
of tissue plasminogen activator (tPA) antigen by immunoadsorption of human
plasma with anti-tPA Ig Sepharose 4B leads to total loss of spontaneous
fibrinolytic activity determined by lysis of a thrombin-induced clot.
Addition of physiological concentrations of purified tPA to tPA-depleted
plasma restores fibrinolytic activity as a function of the length of time
between tPA addition and clotting. Addition of free tPA to tPA-depleted
plasma followed by immediate clotting results in a high rate of
fibrinolysis. In contrast, when free tPA is allowed to incubate in plasma
for 10 to 60 minutes prior to clot formation, the fibrinolytic activity of
tPA is gradually lost. The loss of tPA-induced fibrinolytic activity in
unclotted plasma is accompanied by decreased partitioning of tPA antigen
into fibrin after clotting and is kinetically correlated with the formation
of a 100 kilodalton (kDa) tPA complex as demonstrated by SDS-gel
electrophoresis and fibrin-agar zymography. These results suggest that free
tPA is susceptible to complexation by the plasma inhibitor in the absence
of a clot. Fibrin formation renders tPA relatively inaccessible to
inhibition. The tPA antigen isolated from stored plasma consists mainly of
100 kDa activity in SDS-gel electrophoresis and zymography, indicating that
the tPA complex is resistant to dissociation by SDS. Upon rezymography of
the sliced gel, only a 60 kDa tPA activity is found, suggesting that the
activity at 100 kDa is at least partly due to free tPA dissociated from the
complex during the first zymography. Conversion of tPA complex to
enzymatically active free tPA also occurs with brief SDS exposure followed
by incubation in the presence of excess Triton X-100 or by hydroxylamine
treatment. These results reconcile the apparent discrepancy of the 100 kDA
inhibitor-tPA complex manifesting plasminogen activation activity during
zymography. The plasma tPA- inhibitor complex is precipitated strongly by
antisera against plasminogen activator inhibitors (PAIs) of human Hep G2
hepatoma and HT- 1080 fibrosarcoma cells and weakly by antiserum against
bovine aortic endothelial cell PAI but not by antiserum against a placental
PAI (PAI- 2) suggesting that the plasma inhibitor is immunologically
related to Hep G2, HT-1080 and possibly endothedial cell PAIs. Based on the
above findings, a simple model for the initiation and regulation of plasma
fibrinolysis at the PA level has been formulated.
Volume 69,
Issue 5,
pp. 1354-1362,
05/01/1987
Copyright © 1987 by The American Society of Hematology
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